Process for the production of 7-acylamidocephalosporins

ABSTRACT

New 7-diacylimido cephalosporins are produced by reacting 7-acylamido cephalosporins with an acylating agent in the presence of a silyl reagent. The 7-diacylimido cephalosporins are cleaved to produce cephalosporins containing a different acylamido moiety. Thus, the aminoadipoyl group of cephalosporins produced by fermentation is replaced by a different acyl group by the process of the present invention. The cephalosporins produced are antibiotics having enhanced activity against gram-negative and gram-positive pathogens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 356,873, filed May 3, 1973,now abandoned, which is a continuation-in-part of copending application,U.S. Ser. No. 149,364, filed June 2, 1971 and of copending application,U.S. Ser. No. 223,005, abandoned filed Feb. 2, 1972; which in turn was acontinuation-in-part of copending application, U.S. Ser. No. 149,364,filed June 2, 1971.

BACKGROUND OF THE INVENTION

One method of producing cephalosporins used medicinally as antibioticscomprises converting cephalosporin C to 7-aminocephalosporanic acid andthen acylating this product to produce the desired7-acylamidocephalosporin. This method suffers from the disadvantage thatit is necessary to first isolate and purify the intermediate7-aminocephalosporanic acid. Accordingly, other methods have been soughtwhich would avoid the need of preparing the 7-aminocephalosporanic acid.

More recently it has also been found that cephalosporins having amethoxy substituent in place of the hydrogen substituent at C-7 areproduced by various microorganisms. These cephalosporins likewisecontain an aminoadipoyl group, and it is desired to replace thisaminoadipoyl group with other acyl groups in order to provide newcephalosporins of enhanced antibiotic activity.

SUMMARY OF THE INVENTION

In accordance with the present invention, we have found thatcephalosporin compounds can be transacylated as follows: ##STR1## inwhich B' and R' represent acyl groups, R₁ represents hydrogen or asubstituent such as methoxy, R" represents hydrogen or a blocking groupand A represents hydrogen or an organo substituent unaffected during thedescribed reactions or reconvertible thereto by the removal of anyblocking or protecting groups.

Thus, in the above flowsheet the cephalosporin compound I is reactedwith an acylating agent in the presence of a silyl reagent to producethe 7-diacylamido cephalosporin compound (II) which is then cleaved toproduce the new 7-acylamido cephalosporin compound (III).

The step of producing the diacylated product is best effected byintimately contacting the cephalosporin compound with an acylating agentin a suitable solvent medium in the presence of the silyl reagent. Thetemperature at which the reaction is carried out is not particularlycritical and temperatures from about -20° C. to about 100° C. aregenerally satisfactory, although we prefer to carry out the reaction attemperatures from about 25° C. to 70° C. Solvents which do not containan active hydrogen such as chloroform, acetonitrile, methylene chloride,dioxane, benzene, halobenzene, carbon tetrachloride, diethylether, andthe like are suitable mediums for carrying out this reaction.

The acylating agent can be an acyl halide, an anhydride, or a mixedanhydride although generally it is preferred to use an acyl halide, forexample an acyl chloride, as the acylating agent.

Silicon-nitrogen compounds are useful as silyl reagents in carrying outthe processes of the present invention. Thus, silicon-nitrogen compoundsof the general formula

    (R.sub.5).sub.n Si-(NHR.sub.10).sub.m

wherein R₅ represents alkyl, aryl or aralkyl groups having 1-8 carbonatoms or substituted alkyl, aryl or aralkyl groups having a cyanosubstituent or halo substituents, n represents 2 or 3, R₁₀ represents agroup containing an acyl group of a carbonic acid, a carboxylic acid,carbamic acid, sulfonic acid, sulfamic acid, sulfinic acid, a phosphonicacid, phosphoric acid, and the like. Examples of these silyl reagentsthat might be mentioned are those of the formulas ##STR2##

In the above formulas, R₅ is as defined above; R₆ represents hydrogen orR₅ ; R₇, R₈ and R₉ represent alkyl, aryl or aralkyl substituentscontaining 1-8 carbon atoms; B represents an alkylene group having 2-5carbon atoms; and D represents B or a monocyclic aryl group. Specificsilylating agents that might be mentioned are trihydrocarbylsilyl amidessuch as N-(trimethylsilyl)acetamide, N-(tripropylsilyl)acetamide,N-(tributylsilyl)acetamide, N-(triphenylsilyl)acetamide orN-(tribenzylsilyl)acetamide, N,O-bis(trimethylsilyl)acetamide,N,O-bis(triphenylsilyl)acetamide, N,O-bis(tribenzylsilyl)acetamide,N-(trimethylsilyl)trifluoroacetamide,N-(tributylsilyl)trifluoroacetamide, N-(trimethylsilyl)benzoamide, asilyl substituted urea such as N-(trimethylsilyl)diphenylurea, aN,N-bis(trimethylsilyl)diphenylurea, a silyl urethane such asN-(trimethylsilyl)ethylcarbamate, a silyl imide such asN-triphenylsuccinimide or N-(trimethylsilyl)phthalimide, a silylsulfonamide such as N-(trimethylsilyl)benzene sulfonamide, a dialkyl,diaryl or a diaralkyl silyl reagent such asbis(ethoxycarbonylamino)dimethyl silane and the like.

The substituent at the 3-position in I, II and III above, namely, --CH₂A, represents hydrogen or various substituents which are well known inthe cephalosporin art. Thus, when A is hydroxy it includes the lactoneformed with the carboxy group at 4, and when A is amino it includes thelactam formed with the carboxy group at 4. The substituent A alsorepresents azido, halo, cyano, alkoxy, aryloxy, aralkyloxy,heterocycleoxy, mercapto, alkylthio, arylthio, aralkylthio,heterocyclethio, amino, alkylamino, alkanoylamino, hydroxyphenyl,acylthio, acyloxy, sulfamoyloxy, and the like. The heterocycles can be a5- or 6-membered hetero ring containing one or more nitrogen, oxygen orsulfur atoms. The acyl group can be a loweralkanoyl group of 2-6 carbonatoms, carbamoyl, or thiocarbamoyl and N-alkyl or N,N-dialkylderivatives thereof. The alkyl group of the foregoing substituentscontains 1-6 carbon atoms and may be further substituted radicals suchas alkoxy, halo, amino, cyano, carboxy, sulfo and the like.

The acyl substituents represented by B and R' in formulas I, II and IIIabove are preferably carboxylic acid radicals. Examples of such radicalsthat might be mentioned are those of the general formula R₁₁ R₁₀ CHCOwherein R₁₀ and R₁₁ are as defined below represent a preferred group ofsubstituents because of their generally useful antibiotic activity. R₁₀represents hydrogen, halo, amino, guanidino, phosphono, hydroxy,tetrazolyl, carboxy, sulfo or sulfamino. R₁₁ represents phenyl,substituted phenyl, a monocyclic heterocyclic 5- or 6-membered ringcontaining one or more oxygen, sulfur or nitrogen atoms in the ring,substituted heterocycles, phenylthio, heterocyclic or substitutedheterocyclic thio groups; or cyano. The substituents can be halo,carboxymethyl, guanidino, guanidinomethyl, carboxaminomethyl,aminomethyl, nitro, methoxy or methyl. Examples of these preferredsubstituents that might be mentioned are phenacetyl,3-bromophenylacetyl, p-aminomethylphenylacetyl,4-carboxylmethylphenylacetyl, 4-carboxamidomethylphenylacetyl,2-furylacetyl, 5-nitrofurylacetyl, 3-furylacetyl, 2-thienylacetyl,5-chlorothienylacetyl, 5-methoxythienylacetyl,α-guanidino-2-thienylacetyl, 3-thienylacetyl, 4-methylthienylacetyl,3-isothiazolylacetyl, 4-methoxyisothiazolylacetyl, 4-isothiazolylacetyl,3-methylisothiazolylacetyl, 5-isothiazolylacetyl,3-chloroisothiazolylacetyl, 3-methyl-1,2,5-oxadiazolylacetyl,1,2,5-thiadiazolyl-4-acetyl, 3-methyl-1,2,5-thiadiazolyl-4-acetyl,3-chloro-1,2,5-thiadiazolyl-4-acetyl,3-methoxy-1,2,5-thiadiazolyl-4-acetyl, phenylthioacetyl,4-pyridylthioacetyl, cyanoacetyl, tetrazolylacetyl,α-fluorophenylacetyl, D-phenylglycyl, 3-hydroxy-D-phenylglycyl,2-thienylglycyl, 3-thienylglycyl, phenylmalonyl, 3-chlorophenylmalonyl,2-thienylmalonyl, 3-thienylmalonyl, α-phosphonophenylacetyl,α-sulfaminophenylacetyl, α-hydroxyphenylacetyl, α-tetrazolylphenylacetyland α-sulfophenylacetyl.

The step of cleaving the original acyl group can be effected in severalways, namely, by prolonging the reaction time, by the addition of analcohol such as a loweralkanol or loweralkyl thiol, or by hydrolysis inan aqueous solution containing a small amount of an acid or a base.Thus, in some cases cleavage is effected by the addition of aloweralkanol or loweralkyl thiol containing from 1-6 carbon atoms, anaralkanol such as benzyl alcohol or a corresponding thiol and with othercompounds the cleavage is effected by aqueous hydrolysis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with a preferred embodiment of the present invention, itis now found that cephalosporins obtained by fermentation can beconverted to derivatives having a different acyl group in place of theaminoadipoyl group without first cleaving this group and thenreacylating the intermediate 7-amino compound. The process of thisinvention, therefore, provides a facile means of producing cephalosporincompounds and valuable intermediate products useful in this process. Thegeneral process is illustrated in the following flowsheet: ##STR3##

In the formulas of the above flowsheet, R₁ represents hydrogen ormethoxy, A represents hydrogen or a substituent unaffected during thedescribed reactions or reconvertible thereto by the removal of anyprotecting or blocking groups, R' represents an acyl group, R"represents hydrogen or a blocking or protecting substituent and R₂represents a blocking or protecting substituent.

In accordance with the above flowsheet, the cephalosporin compound IV ora derivative thereof wherein the amino substituent and/or the carboxygroups are blocked or protected (V) is reacted with an acylating agentin the presence of a silyl compound to produce the intermediatediacylated product (VI or VII). The aminoadipoyl moiety of the latterproduct is then selectively cleaved to produce the new acylatedcephalosporin compound (VIII), or a salt thereof when R" is hydrogen.

Although the process of our invention can be carried out withoutblocking or protecting the amino and carboxy groups of the startingcephalosporin compound, we generally prefer to carry it out by firstblocking or protecting both the amino and carboxy groups since maximumyields of the desired new cephalosporin compound are obtained with suchprotected compounds.

An illustrative, more detailed description of this preferred process ofour invention is shown in the following flowsheet: ##STR4## wherein R₁represents hydrogen or methoxy, R₂ and R₃ represent blocking orprotecting groups, A' represents an acyl group and R' represents an acylgroup.

In accordance with this process, the amino group of the startingcephalosporin compound (IX) is first blocked by reaction with a suitablereagent to protect the 5'-amino-substituent. Thus, the amino group isblocked by amino protecting groups such as acyl, aroyl, alkoxycarbonyl,alkylsulfonyl, arylsulfonyl, and the like in accordance with methodswell known in this art. Specific groups suitable for blocking the aminogroup that might be mentioned are trichloroethoxycarbonyl, tertiarybutoxycarbonyl, benzoylmethoxycarbonyl, trimethylsilyl,p-methoxybenzyloxy, 2-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl,chloroacetamido, o-nitrophenylthio and the like, although we generallyprefer to utilize the trichloroethoxycarbonyl derivative which isconveniently prepared by reacting the cephalosporin compound withtrichloroethoxycarbonyl chloride in the presence of an acid scavengersuch as a base of a basic salt, for example a dialkyl phosphate, and thelike.

It is generally preferred to carry out the above-described reactionswith a cephalosporin compound wherein the carboxy group is likewiseblocked or protected (XI) since maximum yields of the desired productare obtained with such derivatives. The blocking or protecting group ispreferably one which can be removed to obtain the free acid withoutdisruption of the β-lactam group since the cephalosporin compounds areusually used in the form of salts such as alkali metal salts or an aminesalt. Protecting groups suitable for this purpose are well known in thisart. Examples of suitable derivatives that might be mentioned are estersof alcohols, phenols, mercaptans, and thiophenols of the general formula--COXR₄ wherein R₄ represents the radical of an alcohol or a thiol suchas methyl, ethyl, tertiary butyl, a substituted alkyl such asphthalimidomethyl, succinimidomethyl, phenacyl, a substituted phenacylfor example p-bromophenacyl, a β-substituted ethyl group such as2,2,2-trichloroethyl, 2-methylthioethyl, 2-(p-methylphenyl)ethyl,2-(p-methylphenyl)sulfonylethyl, 2-methylaminoethyl, 2-chloro(orbromo)ethyl, benzyl, a substituted benzyl group such as p-nitrobenzyl,p-methoxybenzyl, 3,5-dinitrobenzyl, 2,4,6-trimethylbenzyl,3,5-dichloro-4-hydroxybenzyl, and the like, a benzhydryl or substitutedbenzhydryl group such as p-methoxybenzhydryl, an acyloxy alkyl groupsuch as acetoxymethyl, pivaloyloxymethyl, an alkoxy group such asmethoxymethyl, or a monocyclic aryl group for example phenyl orsubstituted phenyl such as p-nitrophenyl or 3,5-dinitrophenyl. Theseprotecting or blocking groups for the carboxy substituents are readilyprepared in accordance with processes well known in this art.

The protected cephalosporin compound is then reacted with an acylatingagent in the presence of the silyl compound such as those describedabove to obtain the imide or diacylated product (XII). The acylatingagent can be an acid halide (chloride or bromide), or a functionalequivalent thereof such as an acid anhydride, a mercaptide, a mixed acidanhydride with other carboxylic acids, an activated ester of thecarboxylic acid such as the p-nitrophenyl ester, and the like.

The preferred acylating agents used in the process of the presentinvention are those of carboxylic acids. The preferred acyl groupsrepresenting R' in the above flowsheet are those of the general formula##STR5## wherein X is hydrogen, halogen, amino, guanidino, phosphono,hydroxy, tetrazolyl, carboxy, sulfo, or sulfamino; R₃ is phenyl,substituted phenyl, a monocyclic heterocyclic 5- or 6-membered ringcontaining one or more oxygen, sulfur or nitrogen atoms in the ring,substituted heterocycles, phenylthio, phenyloxy, heterocyclic orsubstituted heterocyclic thio groups, loweralkyl (1-6 carbon atoms), orcyano; the substituents on the R₃ group being halo, carboxymethyl,guanidino, guanidinomethyl, carboxamidomethyl, aminomethyl, nitro,methoxy or methyl.

When the acylating agent contains groups such as amino or carboxy, thesegroups can be blocked or protected during the acylation reaction andlater removed in accordance with methods known in this art.Alternatively, the acylating agent can contain a substituent such asazido which can be reduced to an amino substituent pursuant to knownmethods.

Especially preferred acylating agents that might be mentioned are thosehaving an acetyl or substituted acetyl group such as phenylacetyl,thienylacetyl (2- and 3-thienylacetyl), furylacetyl (2- and3-furylacetyl), phenoxyacetyl, phenylthioacetyl, α-azidophenylacetyl,and the like since the resulting acylated cephalosporin compounds haveenhanced antibiotic activity.

The process of our invention is preferably carried out with a 3-CH₂ OA'cephalosporin compound wherein A' represents an acyl group. Examples ofsuch acyl groups that might be mentioned are loweralkanoyl (C₂ -C₆), andespecially acetyl, and carbamoyl since the cephalosporin compoundshaving such groups have enhanced antibiotic activity. Also, the acetylgroup can be readily replaced to obtain other 3-substitutedcephalosporins pursuant to methods well known in this art.

The silyl reagent is preferably a triorganosilyl or diorganosilylcompound such as those mentioned above. The trialkylsilyl compounds, andespecially the trimethylsilyl compounds which are readily available, areparticularly useful and preferred for this purpose. Examples of suchcompounds are the trialkylsilyl compounds shown in formulas A-M above,examples of which that might be mentioned are N-trimethylsilylacetamide, N-trimethylsilyl trifluoroacetamide,N,O-bis(trimethylsilyl)acetamide,N,O-bis(trimethylsilyl)trifluoroacetamide, N-trimethylsilylbenzenesulfonamide, N-trimethylsilyl methylsulfonamide, N-trimethylsilylurea, N,N-bis(trimethylsilyl)urea, N-trimethylsilyl-N,N'-diphenylurea,N-trimethylsilyl-N,N'-dimethylurea, N-trimethylsilyl ethylurethane,N-trimethylsilyl methylurethane, N-trimethylsilyl benzylurethane,N-trimethylsilyl succinimide, N-trimethylsilyl phthalimide, and othertrialkylsilyl compounds such as the corresponding triethyl compounds.

The step of converting the protected cephalosporin compound (XI) to theimide to diacylated product (XII) is preferably effected by intimatelycontacting the cephalosporin compound with the acylating agent in asuitable solvent medium in the presence of the silyl compound. Thetemperature at which this reaction is carried out is not critical, andtemperatures from about -20° C. to about 100° C. are generallysatisfactory, although we prefer to carry out the reaction attemperatures from about 25° C. to 70° C. Various solvents which do notcontain an active hydrogen such as chloroform, acetonitrile, methylenechloride, dioxane, benzene, halobenzene, carbon tetrachloride anddiethylether are most suitable as mediums for the reaction mixtures.

The step of cleaving the original aminoadipoyl group can be effected inseveral ways, namely, by prolonging the reaction time, by the additionof an alcohol such as a loweralkanol or a loweralkyl thiol or byhydrolysis in an aqueous solution containing a small amount of an acidor a base. Thus, in some cases cleavage is effected by the addition of aloweralkanol or loweralkyl thiol containing from 1-6 carbon atoms, andaralkanol such as benzyl alcohol or the corresponding thiol. During theacylation reaction some cleavage of the aminoadipoyl group occurs,depending upon the conditions under which the acylation is effected.Thus, prolonged heating of the reaction mixture can result in thecleavage of the aminoadipoyl group and the preparation of the desired7-acylated cephalosporin compound.

In accordance with a further embodiment of the present invention, it isfound that the cleavage of the diacylated product or imide is alsoeffected by reacting the imide with a silyl halide such astrimethylsilyl chloride. This process is readily carried out by heatingthe trimethylsilyl chloride with the imide in a suitable non-reactivesolvent such as ethylene dichloride at about 60° C. for about 1 hour.The desired monoacylated cephalosporin compound is then recovered fromthe reaction mixture in accordance with procedures known in this art.

Alternatively, pursuant to another embodiment of this invention, it isfound that during the acylation process spontaneous cleavage of theimide occurs depending upon the nature of the silyl reagent used. Thus,when an ester of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid is reacted with 2-thienylacetyl chloride in the presence ofN-trimethylsilyl benzene sulfonamide at 65° C. for about 10 hours, thecorresponding ester of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid is isolated directly from the reaction mixture in good yield.

When the protecting group of the amino substituent of the aminoadipoylmoiety such as a trichloroethoxycarbonyl or a t-butoxycarbonyl group isremoved by suitable means, a selective cleavage of the aminoadipoylgroup occurs. This removal of the protecting group of the amino functionapparently results in an internal cyclization of the aminoadipoyl groupresulting in cleavage of the group as the α-carboxylic ester of theformula ##STR6##

Our present evidence indicates that this is the mechanism of thiscleavage; however, we do not wish to be bound by this explanation of howthe cleavage occurs since subsequent studies may establish that theproduct is cleaved and extruded in some other manner. This explanationof how the cleavage occurs is presented to provide a betterunderstanding of our invention.

The cleavage of the protective groups on the amino and carboxy functionsis accomplished in accordance with procedures well known in this art.Thus, for example, the trichloroethoxycarbonyl group is removed byreaction with zinc and acetic acid, and the t-butoxycarbonyl andbenzhydryl groups are removed by reaction with trifluoroacetic acid.

In accordance with a further aspect of our invention, new 7-diacylamidocompounds obtained by our process are not only useful as intermediatesin the preparation of the monoacylated cephalosporins but are usefulantimicrobial products active against various pathogenic microorganisms.

EXAMPLE 13-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

Step A

7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

The mono-sodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (20.5 gm.) is dissolved in the mixture of acetone (80 ml.) andaqueous 10% dipotassium hydrogen phosphate (240 ml.). To this solutionis added dropwise trichloroethoxycarbonyl chloride (25 gm., 118 mmoles)in acetone (80 ml.). During the addition the pH of the solution is keptat 9.1 by gradual addition of 2.5 N sodium hydroxide solution. After 30minutes the mixture is extracted with ethyl acetate, the ethyl acetatelayer discarded, and the aqueous layer is acidified to pH 2.5 withconcentrated hydrochloric acid. The precipitated product is extractedinto ethyl acetate. After drying over sodium sulfate and removing thesolvent in vacuo, the title compound is obtained as an oil.

Uv: (ch₃ oh) λmax. 262.5 ε=5450

Nmr: (solvent -- DMSO, d₆) δ=3.43 (O--CH₃, s), 4.73 (2--H₂, partiallyvisible), 4.81 ##STR7## 5.12 (6-H, s), ˜4.74 (10--H₂, partiallyvisible). Step B

Di-benzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

To the solution of the above7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in ethyl acetate (500 ml.) is added diphenyldiazomethane (17 gm.)in 200 ml. of ether. After agitating the mixture overnight, it isextracted successively with sodium bicarbonate and sodium chloridesolutions. The solvent is evaporated from the dried solution to afford acrude product which is purified by chromatography on silica gel. A 2:1mixture of chloroform and ethyl acetate is used for elution. Thismaterial showed a single spot on TLC chromatography.

Uv: (ch₃ oh) λmax. 2650μm ε7000

Nmr: (solvent -- CDCl₃) δ=3.45 (O-CH₃, s), 3.35 (2--H₂, partiallyvisible, 4.69 ##STR8## 5.03 (6-H, s), ˜4.88 (10--H₂, partially visible).Step C

Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of the di-benzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (1.1 gm., 1.18 mmole), acetonitrile (5 ml.) and bis-trimethylsilyltrifluoroacetamide (3 ml.) is allowed to stand at room temperature for 6hours. After this period, the volatile products are removed in highvacuum and the residue is dissolved in 3 ml. of methylene chloride. Tothis solution is added phenylacetyl chloride (0.23 ml., 1.79 mmole) andthe mixture is allowed to stand at room temperature for 65 hours. Afterthis, the solution is evaporated and the residue is dissolved in 5 ml.tetrahydrofuran and 0.7 ml. of 2.5 N hydrochloric acid. After 20 minutesreaction time the solvent is evaporated and the residue is partitionedbetween methylene chloride and sodium bicarbonate solution. The organiclayer is washed with sodium chloride solution, dried and evaporated todryness. The crude product thus obtained is purified by chromatographyon silica gel, using chloroform ethyl acetate 95:5 as eluant. The titlecompound obtained appears homogenous on thin layer chromatography.

Uv: (ch₃ oh) λmax. 2640μm ε6650

Nmr: (solvent -- CDCl₃) δ=3.50 (O-CH₃, s), 3.31 (2--H₂, partiallyvisible), 4.67 ##STR9## 5.04 (6-H, s), ˜4.96 (10--H₂, partiallyvisible), 3.95 (13--H₂, s).

Step D

Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

The solution of di-benzyhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (104 mg.) in 90% acetic acid-water (1 ml.) is agitated with 100 mg.of zinc dust for 5 hours. After this, the solution is filtered and thesolvent is removed in vacuo. The residue is partitioned betweenmethylene chloride and water, and the methylene chloride layer isextracted with sodium bicarbonate and sodium chloride solutions. Afterdrying and evaporation a crude product is obtained which is purified bythin layer chromatography utilizing silica gel plates and a 3:2 mixtureof chloroform and ethyl acetate. The product is characterized by its IRand NMR spectra.

Ir: (chcl₃) 1780, 1730 and 1680 cm⁻ ¹

Uv: (ch₃ oh) λmax. 2640μm ε5870

Nmr: (solvent - CDCl₃) δ=3.40 (O-CH₃, s), 3.33 (2-H₂, partiallyvisible), 5.01 (6-H, s), ˜4.88 (10-H₂, partially visible), 3.60 (13-H₂,s).

Step E

3-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicacid (17 mg.) is dissolved in anisole (0.2 ml.) and treated withtrifluoroacetic acid (0.5 ml.) for 5 minutes. After this period, themixture is concentrated rapidly in high vacuum and diluted with ethylacetate. The product is removed from the ethyl acetate solution byextraction with a pH 7.5 sodium phosphate buffer. The buffer solution isacidified to pH 2.5 with dilute hydrochloric acid and the title compoundis removed by extraction with ethyl acetate. After drying andevaporating the solution, the product is obtained. An analytical sampleis obtained by recrystallization from ethylacetate. MP: 159°-161° C.

Uv: (pH 7 buffer) λmax. 2670μm ε8650. IR: (CH₃ CN) 1780, 1735 and 1700.NMR: (solvent - CD₃ CN + D₂ O) δ= 3.42 (O-CH₃, s), 3.35 (2-H₂, partiallyvisible), 5.01 (6-H, s), 4.83 (10-H₂, d), 3.61 (13-H₂, s).

Elemental analysis for C₁₈ H₁₉ O₇ N₃ S: Calc. - C, 51.29; H, 4.54;Found - C, 51.47; H, 4.73.

Two milligrams of the above acid is dissolved in 1 drop of methanol andtreated with a solution of 2 mg. dibenzyl ethylenediamine diacetate inethyl acetate. The dibenzyl ethylenediamine salt of the title compoundprecipitates after standing in the form of needle-like crystals. MP:140°-143° C. UV: (CH₃ OH) λmax. 263μm ε8600.

The starting material, the monosodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid, used in the foregoing example is prepared as follows:

Preparation of Monosodium Salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid Modified Fermentation Process

Step 1

Slants

A lyophilized tube of Streptomyces lactamdurans culture (NRRL 3802) wasopened asceptically and the organism transferred to a medium of thefollowing composition:

Medium XI

1% blackstrap Molasses

1% National Brewer's Yeast

2.5% Difco agar pH 7.0

Water to volume

The slants are inoculated for 7 days at 28° C. When stored in the cold,the slants are stable for more than 13 weeks.

Step 2: Seed Stages: Two Stage System

First Seed: The first seed is inoculated directly from the slant of Step1 to 40 ml. of 1% Primary Dried Yeast N.F., pH 7.0 (obtained from theYeast Product Corporation) in a 250 ml. baffled Erlenmeyer flask. Theflasks were then shaken on a 220 rpm. rotary shaker with a 2 inch throwat 28° C. for a period of from 2 to 3 days.

Second Seed: A 2.5% inoculum from the first seed stage was added to aflask containing a 2% Fleischmann S-150 yeast autolysate, pH 7.0. Thegrowth in this stage is characteristically light and the incubation,performed as in the first stage, was not extended beyond 48 hours.

Step 3: Production Medium

The production medium contains per liter of distilled water 30 g.distiller's solubles, 7.5 g. of primary dried yeast N.F. and 0.25% v/vof an emulsified petroleum product (Mobilpar-S)defoamer. The medium isadjusted to pH 7.0 with a small amount of concentrated sodium hydroxidesolution dispensed into Erlenmeyer flasks and autoclaved for 15 or 20minutes at 121° C. After cooling the medium received a 2.5% inoculum ofthe seed obtained in Step 2. The time of incubation can vary from about50 to 100 hours but an incubation period of about 72 hours is preferred.The volume of media in each flask can vary from 30 to 50 ml. but 40 ml.was used routinely. The level of inoculum can vary from 1 to 5%; but, inpractice, a 2.5% level is generally employed.

Step 4: Assay

When the fermentation was complete, the cells were removed bycentrifugation and the broth was diluted with phosphate buffer, pH 7.0.The concentration of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in the fermentation broth was determined by the standardbiological-disc assay method. The assay organism employed was Vibriopercolans (ATCC 8461). Filter paper discs are immersed into the dilutedbroths and placed on the surface of agar-containing Petri dishes whichwere inoculated with the assay organism Vibrio percolans (ATCC 8461).Also placed on these Petri dishes are discs that had been dippedpreviously in standard solutions containing known concentrations of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid. The discs were incubated overnight at 28° C. and the diameters ofthe zones of inhibition recorded. The concentration of product and thefermented broth is calculated by interpolation from the standard curvewhich relates zone diameter with the known concentrations of standardsolutions of the product. By this procedure it was calculated thatStreptomyces lactamdurans NRRL-3802 produced 78.6 μg/ml. of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in the modified fermentation process.

Step 5: Isolation

The filtered broth is adjusted to pH 7.0 with dilute hydrochloric acidand 2900 ml. is passed through a column containing a strongly basicanion exchange resin (100 g.) having a styrene-divinylbenzene matrix(Dowex 1 × 2 chloride cycle resin) at 10 ml./minute. The spent solventis collected in 500 ml. fractions. The resin column is washed with waterand eluted with 3% ammonium chloride in 90% methanol. The eluate iscollected in 100 ml. fractions. The spent fractions are combined, the pHadjusted to pH 7.2 to 8.0 with dilute sodium hydroxide and adsorbed on astrongly basic anion exchange resin (100 g.) having astyrene-divinylbenzene matrix (Dowex 1 × 2 chloride cycle resin) at 14ml/minute. The column is washed with water and eluted with 5% aqueoussodium chloride. The eluate is collected in 50 ml. fractions andconcentrated. The concentrate is diluted to 500 ml., adjusted from pH8.8 to pH 2.0 with dilute hydrochloric acid and adsorbed on 25 ml. of astrongly acidic cation exchange resin of the sulfonate type having astyrene-divinylbenzene matrix (Dowex 50 × 2 hydrogen cycle resin) at 2.5ml./minute. The column is washed with 25 ml. of water then eluted with2% pyridine until the pH of the column effluent rose to pH 7 (54 ml.).The eluate thus obtained is adjusted to pH 8.0 with dilute sodiumhydroxide and concentrated under vacuum to remove the pyridine andafford the monosodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid.

Elemental analysis for C₁₆ H.sub. N₄ SO₉ Na: Calc.: C, 41.0%; H, 4.5%;N, 12.0%; S, 6.8%; Found: C, 39.31%; H, 4.76%; N, 11.16%; S, 6.46%.

EXAMPLE 23-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

Step A

Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A solution of the di-benzhydryl ester of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (9.3 gm., 10 mmoles), N-trimethylsilyl phthalimide (7.8 gm., 40mmoles) and phenylacetyl chloride (5.3 ml., 40 mmoles) in 50 ml. ofacetonitrile is heated to 40° C. for 20 hours. After this period themixture is cooled to room temperature and filtered. The filtrate isevaporated to dryness and triturated with hexane. The insoluble residue,containing di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid, is used without purification in the next step.

Step B

Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

The crude product from Step A is dissolved in a mixture of ethylacetate(50 ml.), acetic acid (45 ml.) and water (5 ml.). To this solution isadded 20 gm. of zinc powder and the mixture is agitated at roomtemperature for 4 hours. After this, the excess zinc is removed byfiltration and the filtrate partitioned between ethylacetate and water.The organic layer is washed with a sodium bicarbonate solution andwater, dried and the solvent is evaporated. The crude product thusobtained is purified by chromatography on 1 kg. of silica gel, using amixture of chloroform, hexane, and methanol (47:47:6) for elution. Theproduct obtained has the physical characteristics described in Example1, Step E.

Step C

3-Carbamoyloxymethyl-7-methoxy-7-phenylacetamido-3-cephem-4-carboxylicAcid

The title compound is prepared by the procedure described in Example 1,Step F, and has the same physical characteristics as the product ofExample 1.

EXAMPLE 33-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

Step A

Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-2-thienylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of 6.0 gm. (6.3 mmole) of the dibenzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid, 4.7 gm. (40 mmoles) N-trimethylsilyl trifluoroacetamide, 3.42 ml.(25 mmoles) 2-thienylacetylchloride, and 50 ml. of chloroform is warmedat 47° C. for 16 hours. After the solvent is removed by evaporation, thecrude reaction mixture is extracted with hexane, and further purified bychromatography on 1 kg. of silica gel using 10% ethylacetate inchloroform as the eluant.

Uv: (ch₃ oh) λmax. 265μm ε5810

Nmr: (solvent - CDCl₃) δ=3.53 (--OCH₃, s), ˜3.4 (2-H₂, d), 4.74##STR10## 5.05 (6--H, s), ˜5.0 (10-H₂, partially visible), 4.15 (13-H₂,s).

Step B

Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

The di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-3-thienylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (4.2 gm., 3.8 mmoles) is dissolved in 30 ml. of ethylacetate andadded to 30 ml. of 90% aqueous acetic acid and 12 gm. of zinc dust. Themixture is stirred vigorously for 51/2 hours at room temperature. Afterthe zinc is filtered off, excess acetic acid is removed by washing theethylacetate solution with water. The title compound is isolated in thesame manner as described in Example 1, Step E. It is characterized byTLC (7% CH₃ OH in 1:1 CHCl₃ :n-hexane) as a single spot material.

Ir: (chcl₃) 1740, 1800 cm⁻ ¹ ;

Uv: λmax. 263μm ε5800

Nmr: (solvent - CDCl₃) δ=3.45 (--OCH₃, s), ˜3.4 (2-H₂, d), 5.02 (6-H,s), ˜4.92 (10-H₂, partially visible), 3.85 (13-H₂, s).

Step C

3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

A cold solution of the benzhydryl ester of3-carbamoyloxymethyl-7-methyl-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid (1.36 gm.) in 10.88 ml. of anisole is stirred with 5.44 ml. oftrifluoroacetic acid at 0° C. for 1/2 hour. The volatiles are removed inhigh vacuum, and the product is recrystallized from ethyl acetate. MP:165°-167° C. ##EQU1## NMR: (Solvent - CD₃ CN + D₂ O) δ=3.48 (--OCH₃, s),˜3.4 (2-H₂, partially visible), 5.05 (6-H, s), 4.91 (10-H₂, d), 3.86(13-H₂, s).

Elemental analysis for C₁₆ H₁₇ N₃ O₇ S₂ : Calc.: C, 44.96; H, 4.01; N,9.83; Found: C, 44.86; H, 3.99; N, 9.21; S, 15.00.

EXAMPLE 43-Carbamoyloxymethyl-7β-(2-furylacetamido)-7-methoxy-3-cephem-4-carboxylicAcid

Step A

Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-2-furylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of the di-benzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (9.3 gm.), bis-(trimethylsilyl)-trifluoroacetamide (7.0 ml.),2-furylacetylchloride (4.7 ml.) and dichloromethane (50 ml.) is warmedat 47° C. for 16 hours. The solvent is removed by evaporation, the crudereaction mixture is extracted with hexane, and the residue is usedwithout purification in the next step.

Nmr: (solvent - CDCl₃) δ=3.48 (--OCH₃, s), 3.08 (2-H₂, d), 4.63##STR11## 5.02 (6-H, s), ˜4.88 (10-H₂, d), 3.72 (13-H₂, s). Step B

Benzhydryl ester of7β-(2-furylacetamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

The di-benzhydryl ester from Step A is reacted with zinc dust and aceticacid following the procedures described in Example 3 Step B. Aftercrystallization from chloroformhexane, the pure product has thefollowing physical characteristics:

Mp: 168°-171° c.

ir: (chcl₃) 1800, 1720, 1700

Uv: λmax. 265μm ε7200

Nmr: (solvent - CD₃ CN) δ=3.43 (--OCH₃, s), 3.39 (2-H₂, partiallyvisible), 5.0 (6-H, s), 4.75 (10-H₂, d), 3.64 (13-H₂, s).

Step C

3-Carbamoyloxymethyl-7-methoxy-7β-(2-furylacetamido)-3-cephem-4-carboxylicAcid

The3-carbamoyloxymethyl-7-methoxy-7β-(2-furylacetamido)-3-cephem-4-carboxylicacid is prepared from the product of Step B following the proceduredescribed in Example 3, Step C. The product, after recrystallizationfrom ethyl acetate, has a melting point of 156°-161° C.

Uv: (pH 7 buffer) λmax. 265μm ε7200

Ir is consistent with the structure.

Nmr: (solvent - CD₃ CN + D₂ O) δ=3.44 (--OCH₃, s), ˜3.38 (2-H₂,partially visible), 5.02 (6H, s), 4.82 (10H₂, d), 3.66 (13-H₂, s).

EXAMPLE 53-Carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylicAcid

Step A

Di-benzhydryl ester of7β-[(D-5-Trichloroethoxycarbonylamino-5-carboxyvaleryl)thiophenoxyamido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

By following substantially the procedure described in Example 4, Step A,and by substituting for the 2-furylacetyl chloride an equimolar quantityof phenylthioacetyl chloride there is obtained di-benzhydryl ester of7β-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)thiophenoxyamido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid.

Nmr: (solvent - CDCl₃) δ=3.33 (--OCH₃, s), ˜3.23 (2-H₂, partiallyvisible), 4.87 ##STR12## 5.0 (6-H, s), 4.87 (10-H₂, u), 3.68 (13-H₂, s).Step B

Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylicAcid

By following substantially the procedure described in Example 4, Step B,and by substituting the di-benzhydryl ester of7β-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)thiophenoxyamido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in place of the di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-2-furylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid there is obtained, after chromatographic purification,substantially pure product which appears as a single spot on thin layerchromatography (TLC). The IR is in accord with the structure.

Uv: λmax. 274μm ε11350

Nmr: (solvent - CDCl₃) δ=3.34 (--OCH₃, s), 3.24 (2-H₂, partiallyvisible), 5.0 (6-H, s), 4.88 (10-H₂, d), 3.68 (13-H₂, s).

Step C

3-Carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylicAcid

The title compound is prepared from the product of Step B abovefollowing the procedure of Example 3, Step C. The product exhibits asingle spot on TLC. MP: 119°-123° C.

Uv: (pH 7 buffer) λmax. 247μm ε10400

Nmr: (solvent - CD₃ CN + D₂ O) δ=3.38 (-OCH₃, s), 3.34 (2-H₂, partiallyvisible), 5.0 (6-H, s), 4.82 (10-H₂, s), 3.71 (13-H₂, s).

EXAMPLE 6

7β-(d,l-α-azidophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

Step A

7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (50.0 g.) is dissolved in a mixture of 1500 ml. aqueous 5%dipotassium hydrogen phosphate and 1000 ml. acetone and adjusted to pH9.5 with 2.5 N sodium hydroxide solution. To this stirred solution isadded tert-butoxycarbonyl azide (50 ml.) and the pH maintained at 9.5over a 20 hour period. The reaction mixture is then extracted with ethylacetate, the ethyl acetate layer discarded, and the aqueous layer iscooled to 0° C., stirred with 1200 ml. of ethylacetate, and acidified topH 2.5 with concentrated hydrochloric acid. The ethyl acetate layer isseparated, dried over sodium sulfate and concentrated in vacuo, and thesolid so obtained may be used without further purification.

Ir: 1790 (β-lactam), 1700

Uv: (pH 7 buffer) λmax. 263 ε6820

Nmr: (solvent - DMSO, d₆) δ=3.30 (--OCH₃, s), 3.42 (2-H₂, partiallyvisible), 5.06 (6-H, s), 4.78 (10-H, d), 1.38 (t-Bu, s).

Step B:

Di-benzhydryl ester of7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

To a solution of7β-(D-5-butoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (15.0 g.) in ethyl acetate (500 ml.) is added diphenyldiazomethane(5.5 g.) in 70 ml. of ether. The reaction mixture is warmed to 40° C.with stirring and after 30 minutes is treated with additionaldiphenyldiazomethane (5.5 g.) in ether (70 ml.). After 3 hours, thesolvent is removed in vacuo and replaced by a mixture of methanol (500ml.) and water (20 ml.). The methanol-water solution is extracted fourtimes with hexane and then evaporated in vacuo. The residue is dissolvedin ethyl acetate, dried over sodium sulfate and evaporated in vacuo toyield the title compound which is used without purification in the nextstep.

Nmr: (solvent - CDCl₃) δ=3.60 (--OCH₃, s), 3.4 (2-H₂, partiallyvisible), 5.10 (6-H, s), 4.95 (10-H, partially visible).

Step C:

Di-benzhydryl ester of7β-[(D-5'-tert-butoxycarbonylamino-5'-carboxyvaleryl)-D,L-α-azidophenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of the di-benzhydryl ester of7β-(D-5'-tertbutoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (10.8 g.), chloroform (100 ml.),bis-(trimethylsilyl)-trifluoroacetamide (16.2 g.) andD,L-α-azido-phenylacetyl chloride is warmed at 45° C. for 16 hours. Themixture is diluted with chloroform (300 ml.), washed with 2% aqueousbicarbonate and saturated aqueous sodium chloride, dried over sodiumsulfate and evaporated to an oil which is purified by precipitating theproduct from a chloroform solution with hexane. The light yellow solidis used in the next step without further purification.

Ir: 1790 (β-lactam, 1, 1735, 2100 (-N₃)

Nmr: (solvent - CDCl₃) δ=3.70 (--OCH₃, s), 3.2 (2-H₂, partiallyvisible).

Step D:

7β-(D,L-α-azidophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A solution of the di-benzhydryl ester of7β-[(D-5'-tertbutoxycarbonylamino-5'-carboxyvaleryl)-D,L-α-azidophenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (13.0 g.) in anisole (13 ml.) is poured into 65 ml. of cold (0° C.)trifluoroacetic acid. After 5 minutes the solution is poured into 1800ml. of stirred, cold (0° C.) ether. The precipitated solid is collectedand distributed between 10% aqueous disodium acid phosphate and ethylacetate. The ethyl acetate layer is discarded and the aqueous layer islayered with fresh ethyl acetate and the stirred mixture brought to pH 2in the cold with 60% aqueous phosphoric acid. The ethyl acetate layer iscollected, washed with saturated aqueous sodium chloride and then driedover sodium sulfate. Volatiles are removed in vacuo to afford the titlecompound.

Uv: .sub.∥max. 264μm ε7537 231μ ε13567 (pH 7 buffer)

Ir: 1760 (β-lactam) 1705, 2105 (-N₃)

Nmr: (solvent - CD₃ CN) δ=3.36 (--OCH₃, s), 3.50 (--OCH₃, s), 3.40(2-H₂, partially visible), 5.06 (6-H, s), 4.86 (10-H, s), 5.15 (13-H,s).

EXAMPLE 77ε-(D,L-α-aminophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A slurry of 1.0 g. of7β-(D,L-α-azidophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in acetic acid (10 ml.) and water (90 ml.) at 0° C. is stirred withzinc dust (5.0 g.) for 10 minutes and filtered. The filtrate is spargedwith hydrogen sulfide, filtered, and the filtrate freeze dried to afforda white solid which is washed with ether and dried in vacuo to affordthe title compound as a white powder.

Uv: (pH 7 buffer) λmax. 264μ ε6525

Ir: 1770 (β-lactam) 2650, 1550 (HN₃ + )

Nmr: (solvent - D₂ O + HCO₃ -- ) δ=3.78 (--OCH₃, s), 3.84 (--OCH₃, s),3.90 (2-H₂, partially visible).

EXAMPLE 8 3-Acetoxymethyl-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

Step A:

7β-(D-5-Trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicAcid

To a solution of7β-(D-5-amino-5-carboxyvaleramido)-3-acetoxymethyl-3-cephem-4-carboxylicacid (2.5 g., 0.53 mole) in acetone (13 ml.) and aqueous 10% dipotassiumhydrogen phosphate (40 ml.) is added dropwise trichloroethoxycarbonylchloride (3.35 g., 0.159 mole). During the addition the pH of thesolution is kept in the range of from 8.5 to 9 by the gradual additionof a 17% aqueous solution of sodium hydroxide. After 30 minutes themixture is washed with ethyl acetate and the aqueous layer is acidifiedto pH 2.5 with concentrated hydrochloric acid. The precipitated productis extracted into ethyl acetate, the solution is dried over sodiumsulfate, filtered and the solvent removed to afford 2.7 g. of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicacid.

Step B:

Dibenzhydryl ester of7-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetoxymethyl-3-cephem-4-carboxylicAcid

To a solution of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicacid in ethyl acetate (30 ml.) is added diphenyl diazomethane (2.0 g.)in ether (25 ml.). The mixture is stirred overnight and the solventremoved to afford 4.0 g. of crude product. The crude product is purifiedby chromatography on silica gel using chloroform as the eluant to afford2.3 g. of substantially pure dibenzhydryl ester of7-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicacid.

Nmr: (solvent - CDCl₃) δ=2.0 (methyl, s), 4.9 (10-H₂, quartet), 3.2(2-H₂, quartet), 4.95 (6-H, d), 5.92 (7-H), 7.0 (benzhydryl protons, 2s).

Step C:

Dibenzhydryl ester of7-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)-2-thienylacetylamino]-3-acetoxymethyl-3-cephem-4-carboxylicAcid

A mixture of the dibenzhydryl ester of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetoxymethyl-3-cephem-4-carboxylicacid (2.0 g., 0.02 mole), N-trimethylsilyl trifluoroacetamide (1.65 g.,0.09 mole), 2-thienylacetyl chloride (1.31 g., 0.0815 mole) andmethylene chloride (6 ml.) is warmed at 40°-45° C. in an oil bath undera nitrogen atmosphere for 20 hours. The reaction mixture is poured intohexane (100 ml.) and filtered through diatomaceous earth. Removal of thesolvent affords the dibenzhydryl ester of7-[D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)-2-thienylacetylamino]-3-acetoxymethyl-3-cephem-4-carboxylicacid.

Step D:

Benzhydryl ester of3-acetoxymethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic Acid

The dibenzhydryl ester of7-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)-2-thienylacetylamino]-3-acetoxymethyl-3-cephem-4-carboxylicacid is dissolved in ethyl acetate (10 ml.) and added to a mixture of90% aqueous acetic acid (10 ml.) and zinc dust (1.0 g.). The mixture isstirred for two hours at room temperature. The reaction mixture isfiltered to remove the zinc. The reaction mixture is washed successivelywith 2 portions of water, a cold sodium bicarbonate solution and thenwith a saturated sodium chloride solution (15.0 ml.). The ethyl acetatesolution is dried over sodium sulfate, filtered and the solvent removedto afford 1.9 g. of crude product which is chromatographed on silica gelusing a mixture of chloroform and ethyl acetate (50:1) as the eluant toafford 0.380 g. of product which, after recrystallization from ethylacetate, has a melting point of 141.5°-143° C.

Uv: (ch₃ oh) λmax. 263 ε7580 Elemental analysis for C₂₉ H₂₆ N₂ O₆ S₂ :Calc: C, 61.91; H, 4.66; N, 4.98; Found: C, 62.14; H, 4.84; N, 4.91.

Step E:

3-(Acetoxy methyl)-7-(2-thienylacetamido)-3-cephem-4-carboxylic Acid

A cold solution of benzhydryl ester of3-acetoxymethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic acid (100mg.) in anisole (1.0 ml.) and trifluoroacetic acid (0.5 ml.) is stirredat 0° C. for 35 minutes. Carbon tetrachloride (50 ml.) is added and thereaction mixture is concentrated to dryness. The residue is trituratedwith hexane. The hexane is removed by decantation and this residue isdissolved in ethyl acetate (10 ml.), concentrated to 1 ml. and diethylether added to afford precipitate. This precipitate is recrystallizedfrom a mixture of diethyl ether and ethyl acetate to afford 0.025 g. of3-(acetoxymethyl)-7-(2-thienylacetamido)-3-cephem-4-carboxylic acid,m.p. 164° C. Mixed melting point with an authentic sample was 163° C.

EXAMPLE 97-(Trifluoroacetyl)amino-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid A. Dibenzhydryl ester of7-[N-trifluoroacetyl-6S'-(trichloroethoxycarbonylamino-5'-carboxy)valeramido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid

A mixture of dibenzhydryl ester of7-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (2.0 g., 2.1 mm.), trifluoroacetyl chloride (4.0 g., 30.0 mm.),methylene chloride (10 ml.) and N-trimethylsilyl trifluoroacetamide (5ml., 32 mm.) is placed in a tube, sealed and allowed to stand at roomtemperature for 21 hours. After this period, the volatile products areremoved under vacuum to give 6.5 g. of crude material. The product,dibenzhydryl ester of7-[N-trifluoroacetyl-6S'-(trichloroethoxycarbonylamino-5'-carboxy)valeramido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid, so obtained is used directly in the following step.

B. Benzhydryl ester of7-(trifluoroacetylamino)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid

A solution of imide obtained in A above (6.5 g.) in anhydrousacetonitrile (40 ml.) is stirred with activated zinc dust (10 g.) andacetic acid (2.0 ml.) for 1 hour. The resulting mixture is filtered andthe zinc pad washed with methylene chloride. The filtrate isconcentrated to dryness and the residue chromatographed on silica gel(60 g.) and eluted with chloroform-hexanemethanol (50:50-2) to give thetitle compound. IR: 1800, 1750 (CH₂ Cl₂). NMR is in accord with thestructure.

C.7-(Trifluoroacetyl)amino-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid

A cold solution (0°-4° C.) of dioxane (0.04 ml.), methylene chloride(0.27 ml.) and trifluoroacetic acid (0.27 ml.) is added to a coldsolution (0°-4° C.) of anisole (0.19 ml.), methylene chloride (0.52 ml.)and the product of B (100 mg.). The reaction is allowed to continue for40 minutes at 0°-4° C. After this period, the reaction is quenched bythe addition of dioxane (1 ml.) and carbon tetrachloride (10 ml.) andthen concentrated to dryness. The residue is triturated with hexane togive the title compound. IR: 1800, 1740 (CH₃ CN). NMR is in accord withthe structure.

EXAMPLE 10 Benzhydryl3-carbamoyloxymethyl-7-methoxy-7-trifluoroacetamido-3-cephem-4-carboxylicacid

A mixture of the dibenzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (5 g.), trifluoroacetic anhydride (1.4 ml.), trimethylsilyltrifluoroacetamide (2.25 g.) and methylene chloride (20 ml.) is heatedto 40° C. for 3 hours. The mixture is poured into hexane and theprecipitate dissolved in acetonitrile. To this solution is added aceticacid (4 ml.) and zinc powder (25 g.) and the mixture is agitated for 3hours. After filtration the solvent is removed and the residuechromatographed on silica gel (100 g.) using a mixture of benzene andethyl acetate for elution. The titled product is obtained as a whitefoam which exhibits the expected NMR spectra.

EXAMPLE 113-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid A. Di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-(D-5-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-cephem-4-carboxylate

To a cold suspension of di(cyclohexylamine)3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylate(9.85 g.) in methylene chloride (dried over molecular sieves, 170 ml.)at 0° C. is added a solution of chloromethyl methyl ether (1.69 g.) in50 ml. of methylene chloride dropwise over 60 minutes with stirring.After the addition, the suspension is stirred at 0° C. for 2 hours. Themixture is filtered through a pad of celite and the pad rinsed withmethylene chloride (30 ml. × 3). The filtrate is evaporated to drynessin vacuo at 5°-10°C. and the residue shaken with 200 ml. methylenechloride and 200 ml. ice water. The organic layer is separated, washedfurther with ice water (200 ml.), cold 0.1% NaHCO₃ solution (200 ml.),water (200 ml.), and 50 ml. aqueous NaCl solution and dried overmagnesium sulfate. After filtering off the drying agent the solvent isremoved in vacuo to give 6.25 g. of product as a glass.

B. Di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7η-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-thienylacetylamino]-3-cephem-4-carboxylate

To a solution of di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-(D-5-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-cephem-4-carboxylatein 80 ml. of sieve dried methylene chloride is added α-pinene (13.5ml.), N-trimethylsilyl ethylurethane (12.1 ml.) and 2-thienylacetylchloride (7.0 ml.). The solution is aged 17 hours under a dry nitrogenatmosphere while gently refluxing in a 50° C. oil bath. The solution isthen cooled to room temperature, treated with 500 ml. of an aqueoussolution containing 10% sodium chloride and 2.5% sodium bicarbonate,methanol (30 ml.) added and the mixture agitated vigorously for 15minutes at ambient temperature. The layers are separated, the aqueouslayer backwashed with methylene chloride (20 ml.), the combinedmethylene chloride layers washed with saturated aqueous sodium chloride(30 ml.), the sodium chloride solution backwashed with methylenechloride (30 ml.) and the combined methylene chloride solutions driedover magnesium sulfate, filtered and the cake washed with 2 × 3 cakevolumes of methylene chloride. The combined methylene chloride solutionand washes are concentrated in vacuo to 50 ml., quenched dropwise ontovigourously stirred ice cold hexane (500 ml.), and after settling 15minutes the hexane layer is decanted from the gummy solid by filteringthrough celite pad. The gummy solid and pad are washed with ≃50 ml.hexane, then the celite pad washed with 50 ml. methylene chloride andthis methylene chloride solution used to dissolve the gummy solid. Themethylene chloride solution is then placed through the hexaneprecipitation again. The product, di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-[D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-thienylacetylamino]-3-cephem-4-carboxylate,is dissolved in 230 ml. of ethyl acetate and used in the next step.

C. Di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylate

The ethyl acetate solution is charged to a 500 ml. 3-neck round bottomflask fitted with a thermometer mechanical stirrer and a nitrogen line.Zinc dust (50.6 g.) is charged and the solution is stirred while 11.5ml. glacial acetic acid are added. After all the reagents are charged,the reaction is run under a nitrogen atmosphere at 25° C. for 1 hour.The zinc is filtered off and washed with ethyl acetate (2 × 75 ml.). Thefiltrate is washed with 2 × 150 ml. of water and once with cold 2.5%sodium bicarbonate (150 ml.) followed by one wash 100 ml. of saturatedsodium chloride. The ethyl acetate solution is dried over sodiumsulfate, filtered and concentrated to obtain the product as a gum.

Into a cooled (0°-4° C.) jacketed column (2.5 cm. inner diameter) ischarged a slurry of 64 g. of silica gel (60-200 mesh) in methylenechloride (200 ml.). The silica gel is allowed to settle and the excessmethylene chloride drained off to the level of the bed. To this ischarged 6.4 g of the residue in a minimum amount of methylene chloride(5-10 ml.), the flask and column walls being washed with methylenechloride (10 ml.) and then the column eluted with 2 liters of 40% ethylacetate-benzene solution taking 60 ml. fractions. The fractions are thenconcentrated to dryness. Fractions 10-18 containing the product areevaporated to obtain 2.9 g. of di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylate.

The product (2.64 g.) is dissolved in methylene chloride (150 ml.) andfiltered. To this solution is added ethanol (20 ml.) and concentrated atroom temperature on a rotary evaporator to a volume of about 20 ml. Anadditional 20 ml. of ethanol is added to this volume and concentrated toabout 30 ml. The solution is seeded and allowed to crystallize to give2.1 g. of pure ester. m.p. = 151-153° C.

D. 3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid

A suspension of the methoxymethyl ester (920 mg.) in 9.2 ml. of methanol(containing 10% concentrated HCl) is stirred at room temperature for 50minutes. The completeness of reaction is ascertained by tlc analysis,using ethyl acetate/benzene 6:4 system. A cold solution of 1.65 g.NaHCO₃ in 40 ml. of water is added cautiously and the clear solutionconcentrated to a volume of 30-35 ml. at room temperature in vacuo. Theaqueous solution is extracted with ethyl acetate to remove neutralimpurities and the organic layer discarded. The aqueous layer is cooledto 0-5° C., covered with ethyl acetate (40 ml.) and the pH adjusted to1.8 with cold 10% HCl with constant stirring. The layers are separatedand the ethyl acetate layer washed 3 × 3 ml. ice water. The aqueouslayer and washing are combined and then re-extracted with 2 × 20 ml.ethyl acetate. The second ethyl acetate extract is washed twice withcold water and combined with the first extract. The solvent isevaporated to dryness and flushed with 10 ml. of benzene-methanol (8:2)and dried to constant weight. Recrystallization of the crude productfrom methanol/ethyl acetate affords 3-carbamoyl-oxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid.

EXAMPLE 12 3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid A.3-Carbamoyloxymethyl-7-methoxy-7β-[D-5-(2-nitrophenylsulfenylamino)-5-carboxyvaleramido]-3-cephem-4-carboxylicacid

The monosodium salt of 3-carbamoyloxymethyl-7-methoxy-7β-(D-5'-amino-5'-carboxyvaleramido)-3-cephem-4-carboxylic acid (0.37g.) is dissolved in 15 ml. of water. Dioxane (30 ml.) is added, followedby enough sodium hydroxide to adjust the pH to 8.5-9.0.2-Nitrophenylsulfenyl chloride (0.55 g.) is added to the reaction over20 minutes simultaneously with aqueous sodium hydroxide, maintaining thepH between 8.5-9.0. After stirring the reaction for an additional 30minutes, 45 ml. of water is added, the mixture filtered, and thefiltrate extracted with 25 ml. of ethyl acetate. The aqueous layer iscooled to 0-5° C., acidified with concentrated hydrochloric acid, andthe product extracted into ethyl acetate. The ethyl acetate layer iswashed with water, dried over sodium sulfate, filtered, and concentratedto give the titled product; weight 0.60 g.

B. Bis-dicyclohexylamine salt of 3-carbamoyloxymethyl-7β[D-5-(2-nitrophenylsulfenylamino)-5-carboxyvaleramido]3-cephem-4-carboxylic acid

3-Carbamoyloxymethyl-7-methoxy-7β-[D-5-(2-nitrophenylsulfenylamino)-5-carboxyvaleramido]-3-cephem-4-carboxylic acid is dissolved in 25 ml.of ethyl acetate, filtered, and the filtrate is treated over 15 minuteswith 0.675 g. of dicyclohexylamine acetate salt dissolved in 100 ml. ofethyl acetate. Precipitates are collected on a funnel, washed with 10ml. of ethyl acetate, followed by 2 × 10 ml. of isopropyl ether, thendried in vacuo to give 0.38 g. of the above-titled salt.

C. Di-methoxymethyl ester of 3-carbamoyloxymethyl-7β[D-5-(2-nitrophenylsulfenylamino)-5-carboxyvaleramido]3-cephem-4-carboxylic acid

To a solution of 0.25 g. of the bis-dicyclohexylamine salt in 5 ml. ofmethylene chloride under nitrogen is added 0.045 ml. of chloromethylmethylether in 1.5 ml. of methylene chloride over 15 minutes whilemaintaining the temperature of the reaction at 5° C. After 4 hours at 0-5° C. the reaction is filtered and the filtrate washed with 2 ml. ofwater containing 25 mgs. of sodium bicarbonate. The methylene chloridelayer is dried over sodium sulfate, filtered, and concentrated to 0.155g. of crude product. The crude diester is dissolved in 0.5 ml. of amixture of ethyl acetate-benzene (1:1) and chromatographed on 0.75 g. ofsilica gel employing the same solvent mixture to give 0.114 g. of thetitled product.

D. Di-methoxymethyl ester of 3-carbamoyloxymethyl-7β[D-5-(2-nitrophenylsulfenylamino)-5-carboxyvaleryl)-(2-thienylacetyl)amino]-3-cephem-4-carboxylicacid

To a solution of 0.345 g. of di-methoxymethyl ester of3-carbamoyloxymethyl-7β-[D-5-(2-nitrophenylsulfenylamino)-5-carboxyvaleramido]-3-cephem-4-carboxylic acid in 3 ml. of chloroform(alcohol free) is added 0.477 ml. of pinene, 0.430 ml. ofN-trimethylsilyl-urethane, and 0.248 ml. of thienylacetyl chloride andthe mixture stirred for 23 hours at room temperature. The reactionmixture is added to 50 ml. of hexane to give a precipitate. The hexaneis decanted and the precipitate washed with 10 ml. of hexane and thendissolved in 3 ml. of dry methylene chloride and reprecipitated byaddition to 50 ml. of hexane to give 0.252 g. of crude product. Thecrude product is chromatographed over 2.5 g. of silica gel employingethyl acetate-benzene (1:1) to give 0.106 g. of the titled product.

E. 3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid

A solution of 10 mg. of the dimethoxymethyl ester prepared in D above in0.15 ml. of methanol is treated with 0.05 ml. of acetic acid and 10 mg.of thioacetamide to give the methoxymethyl ester after 5 minutes. Whenthe reaction time is extended to 30 minutes, the titled product isformed by loss of the methoxymethyl ester grouping.

Other reagents than thioacetamide which are known in the art to cleavethe nitrophenylsulfenyl group such as thiourea, sulfurous acid,thiourethane, sodium thiosulfate, thiophenol, thioglycolic acid,hydrogen sulfide, and ammonium thiocyanate can be used in place ofthioacetamide.

EXAMPLE 13 3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid

When 2,4-dinitrophenylsulfenyl chloride is substituted for2-nitrophenylsulfenyl chloride in Example 12A and the remaining stepsare carried out as in Example 12B, C, D and E above, the same product isobtained. Thus, these processes are carried out as follows:

A solution of 4.41 g. of the monosodium salt of3-carbamoyloxymethyl-7-methoxy-7β-(D-5'-amino-5'-carboxyvaleramido)-3-cephem-4-carboxylic acid in 106 ml. water and 244 ml. of dioxane istreated with 8.4 g. of 2,4 -dinitrophenylsulfenyl chloride at pH 8.5-9.0by addition of sodium hydroxide as necessary. After 1 hour the reactionis diluted with 350 ml. of water, filtered, and the filtrate extractedwith 2 × 175 ml. of ethyl acetate. The aqueous layer is cooled to 0-5°C., 175 ml. of ethyl acetate added, and the pH adjusted to 2.2 withconcentrated HCl. The ethyl acetate layer is washed with water, driedover sodium sulfate, filtered, and concentrated in vacuo to 8.52 g. ofproduct.

The N-(2,4-dinitrophenylsulfenyl) compound (8.5 g.) in 195 ml. of ethylacetate is treated with 6.8 g. of dicyclohexylamine acetate salt in 700ml. of ethyl acetate. After 45 minutes the product salt is collected andwashed with 100 ml. of ethyl acetate, then 3 × 20 ml. of isopropylether. After drying in vacuo the product weighs 8.7 g.

A suspension of 8.0 g. of N-(2,4-dinitrophenylsulfenyl)dicyclohexylamine salt in 128 ml. of methylene chloride is treated at 0°C. over 25 minutes with 1.5 ml. of chloromethyl methyl ether in 51 ml.of methylene chloride. After 2 hours the reaction mixture is filteredand the filtrate washed with 200 ml. of ice water, 200 ml. of coldsaturated sodium bicarbonate, 2 × 200 ml. of ice water, 200 ml. of coldsaturated sodium chloride, then dried over sodium sulfate, filtered, andconcentrated to 5.14 g. of crude product ester. The product is dissolvedin 25 ml. of ethyl acetate and filtered through a 52 g. of silica gelbed, then concentrated in vacuo to afford 4.72 g. of purified ester.

A solution of 0.50 g. of N-(2,4-dinitrophenylsulfenyl)dimethoxy methylester in 4 ml. of methylene chloride is treated with 0.652 ml. ofα-pinene, 0.586 ml. of N-trimethylsilyl-urethane, and 0.338 ml. ofthienylacetyl chloride at reflux temperature for 17 hours, after whichthe reaction is poured into 50 ml. of ice cold hexane to give a solidwhich is re-precipitated from ice cold hexane to give 0.606 g. of thediacylated product.

20.1 Mg. of the diacylated product in 0.47 ml. of acetic acid is treatedwith 23.2 mg. of thiourea for 95 minutes to give3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid directly as observed by thin layerchromatography comparison with an authentic sample of the same product.

EXAMPLE 14 3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid A. Di-benzyloxymethyl ester of3-carbamoyloxymethyl-7- methoxy-7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-cephem-4-carboxylic acid

A suspension of 985 mg. of the bis-dicyclohexylamine salt of3-carbamoyloxymethyl-7-methoxy-7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-cephem-4 -carboxylic acid in 17 ml. of methylenechloride at 0-1° C. is treated with 0.294 ml. of chloromethyl benzylether in 5 ml. of methylene chloride by addition over 52 minutes. After4 hours the reaction is filtered and the filtrate treated with 2 × 5 ml.of cold saturated sodium bicarbonate, 2 × 10 ml. of ice water, thendried over sodium sulfate and filtered. The filtrate is concentrated toa residue, then slurried with 15 ml. of isopropyl ether and the titledproduct as a solid collected on a funnel (726 mg.).

B. Di-benzyloxymethyl ester of 3-carbamoyloxymethyl-7-methoxy-7β-[D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl-(2-thienylacetyl)amino]-3-cephem-4-carboxylicacid

A solution of 0.20 g. of the di-benzyloxymethyl ester in 1.6 ml. ofmethylene chloride is treated with 0.222 ml. of α-pinene, 0.20 ml. ofN-trimethylsilylurethane, and 0.115 ml. of 2-thienylacetyl chloride atreflux temperature for 23 hours. The reaction mixture is extracted with2 ml. of an ice cold solution containing 2.5% sodium bicarbonate and 10%sodium chloride and the methylene chloride solution washed with 2 ml. ofsaturated sodium chloride solution, dried over magnesium sulfate,filtered, and the filtrate added to 50 ml. of cold hexane to give thetitled product as a precipitate; weight 0.161 g.

C. Benzyloxymethyl 3-carbamoyloxymethyl-7-methoxy-7β(2-thienylacetamido)-3-cephem-4-carboxylate

A solution of the imide prepared in B above (0.150 g.) in 4.1 ml. ofdioxane containing 0.20 ml. of acetic acid is treated with 0.86 g. ofactivated zinc for 2 1/2 hours. The reaction is filtered and the residueon the funnel washed with 20 ml. of chloroform. The combined filtrate iswashed with 2 × 20 ml. of water, 10 ml. of cold saturated sodiumbicarbonate, 10 ml. of saturated sodium chloride, then dried overmagnesium sulfate, filtered, and concentrated to give 0.143 g. ofresidue containing the benzyloxymethyl ester.

D. 3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid

25.4 Mg. of the benzyloxymethyl ester is dissolved in 0.25 ml. of asolution made from 9 ml. of methanol and 1 ml. of concentratedhydrochloric acid. After 1 hour the reaction is complete and the productis isolated by concentration in vacuo.

EXAMPLE 15 Methoxymethyl ester of 3-Carbamoyloxymethyl-7-methoxy-7β(2-thienylacetamido)-3-cephem-4-carboxylic acid A.7-(D-5-chloroacetamidoadipamido)-7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid

The monosodium salt of 7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4 -carboxylic acid (14.2 g.,63.6% pure) is dissolved in 200 ml. of an ice cold aqueous solution of4.9 ml. of 85% H₃ PO₄ (adjusted to pH 7.0 with 50% NaOH) contained in acold jacketed Waring blender. A pH electrode is suspended in thesolution and, with maximum agitation, the pH is adjusted to 11.0. A 4.0ml. aliquot of freshly distilled chloroacetyl chloride is added and thepH maintained at 10.5-11.0 by NaOH addition. Additional acid chloride(4.0 ml., then 2.0 ml.) is charged in the same manner and after 10minutes maximum agitation the conversion to the title compound is judgedcomplete by thin layer chromatography.

The reaction mixture is stirred with an equal volume of ethyl acetateand acidified with concentrated HCl to pH 1.7 at 0° C. The layers areseparated and the extraction repeated with four additional volumes ofethyl acetate containing 1 ml. each of acetic acid. The combinedextracts are dried over Na₂ SO₄, concentrated in vacuo and the yellow,oily residue precipitated from 300 ml. of benzene. The crude titlecompound (5.35 g., 51%, ca 60% pure) is collected by centrifugation.

B. Bis-methoxymethyl ester of 3-carbamoyloxymethyl-7-methoxy-7-(D-5-chloroacetamidoadipamido)-3-cephem-4- carboxylic acid

To 100 ml. of sieve dried methylene chloride is added 1.35 ml. ofsym-collidine and 2.68 g. (5.13 mmol) of the product obtained in Aabove. The light yellow suspension is cooled to 5° C. under N₂ and two0.335 ml. portions of ClCH₂ OCH₃ are added with rapid stirring andmaintenance of the reaction temperature at 5-10° C. The mixture isstirred 45 minutes and 10% more of collidine and ClCH₂ OCH₃ added. Aftera total of 1.75 hours the mixture is filtered. The filtrate is washedwith three 50 ml. portions of ice water, 50 ml. of saturated NaClsolution and dried over Na₂ SO₄. The filtered extracts are concentratedin vacuo giving 1.80 g. of crude diester which is purified bychromatography on 30 g. of coarse silica gel. Elution with 400-500 ml.of CH₂ Cl₂ gives by-products and elution with 500 ml. of ethyl acetategives 1.22 g. of white, amorphous, solid title diester.

C. Methoxymethyl 3-carbamoyloxymethyl-7-methoxy-7β-[D-5-chloroacetamidoadipoyl-(2-thienylacetyl)amino]3-cephem-4-carboxylate

To 100 mg. of the product of B above in a sealed Hypovial is added 0.350ml. of ethanol-free CHCl₃, 0.068 ml. of N-trimethylsilyl-urethane (0.395mmol), and 2-thienylacetyl chloride (0.034 ml., 0.274 mmol). Thehomogeneous mixture is heated 4 hours at 50° C. and then allowed tostand 24 hours at room temperature. The reaction mixture is precipitatedfrom 3 volumes of dry hexane and the precipitate collected bycentrifugation, dissolved in CH₂ Cl₂, and the solution concentrated invacuo to a light yellow foam; yield 80 mg. Thin layer chromatography andan nmr spectrum shows this material to be essentially pure titlecompound.

D. Methoxymethyl ester of 3-carbamoyloxymethyl-7-methoxy7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid

The crude imide obtained in C above (20 mg.) in 0.250 ml. of absoluteethanol is treated with 5 mg. of thiourea for 24 hours at roomtemperature. The methoxymethyl ester is shown to be present by thinlayer chromatography and the starting imide has completely reacted.

Alternatively, the crude imide (25 mg.) and zinc dust (100 mg.) aretreated with a mixture of 0.5 ml. of ethyl acetate and 0.025 ml. ofglacial acetic acid. After 4 hours at room temperature the presence ofthe titled product is demonstrated by thin layer chromatography.

EXAMPLE 163-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid

One-tenth g. of bis-dicyclohexylamine salt of3-carbamoyloxymethyl-7β-[D-5-(2,4-dinitrophenylsulfenylamino)-5-carboxyvaleramido]-3-cephem-4-carboxylicacid is placed in methylene chloride (3 ml.) and stirred at 25° C. for 2hours with 26.5 μl. of trimethylsilyl chloride. Di-cyclohexylaminehydrochloride (42 mg.) is filtered off and the filtrate treated with 100μl. of N-trimethylsilyl ethyl carbamate and 43.5 μl. (0.35 mmole) of2-thienylacetyl chloride. The solution is refluxed for 19 hours. Six ml.hexane is added, the mixture stirred, and the supernatent decanted. Thisis repeated twice more to remove excess 2-thienylacetyl chloride. 85 Mg.of hexane insoluble material is treated with 50 mg. of thioacetamide in1 ml. methanol and stirred for 2.5 hours. Ethyl acetate (5 ml.) is addedand washed with sodium bicarbonate solution (pH 8.3). The water layer iswashed with ethyl acetate and the aqueous layer acidified to pH 2.8 with17% H₃ PO₄. Product (28 mg.) containing the titled product is extractedfrom the acidic water with ethyl acetate.

EXAMPLE 17 Methoxymethyl3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylate

To a mixture of 6.75 ml. of α-pinene and 12 ml. of dichloroethane isadded 5.55 mg. of N-trimethylsilyl benzene sulfonamide, 3.5 ml. of2-thienylacetyl chloride, and 500 mg. of methoxymethyl7β-(D-5-trichloroethoxy-carbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylate.The resulting reaction mixture is heated under reflux at 65° C. for 10hours. 3.4 Ml. of the resulting solution is then cooled in dry ice andthen warmed to reflux temperature. The resulting solution containingsolid crystals is diluted to 100 ml. with dichloroethane. Assay of theresulting solution indicates the presence of 41.8 mg. of theabove-titled product.

EXAMPLE 183-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid

To a slurry of 200 mg. of7β-(D-5'-t-butoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in ethanol-free chloroform (2 ml.) is added 432 mg. ofN-trimethylsilyl trifluoroacetamide and 0.56 mg. of triethylamine. Tothis mixture is added 182 μl. of thienylacetyl chloride. The solution isheated in an oil bath to 45° C. for 91/2 hours. The mixture is thenquenched by adding n-hexane. The solution is centrifuged and the hexanepoured off the gummy solid. This procedure is repeated and the hexanefractions discarded. The gummy solid is dissolved in 10 ml. formic acidfor 30 minutes. The formic acid is removed in vacuo. The solid isdistributed between ethyl acetate (3 ml.), water (3 ml.) and the pHadjusted to 2.5. The ethyl acetate layer is concentrated to dryness.Both TLC analysis and liquid chromatography show the presence of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid. The crude product is purified by preparative TLC using silica gelplates and a solvent consisting of benzene 50; methanol 10; acetic acid6.

EXAMPLE 193-Carbamoyloxyethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid

To a slurry of 200 mg. of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in 2 ml. of ethanol-free chloroform is added 430 mg. ofN-trimethylsilyl benzenesulfonamide and 0.6 mg. of triethylamine. Tothis mixture is added 182 μl. of thienylacetyl chloride. The resultingreaction mixture is heated in an oil bath at 45° C. for 10 hours andthen quenched by adding to n-hexane. The solution is centrifuged and thehexane discarded from the gummy solid. This procedure is repeated twicemore and the hexane fractions are discarded. The gummy solid isdistributed between 3 ml. of ethyl acetate and 3 ml. of water, and thepH is adjusted to 2.5. Concentration of the ethyl acetate layer todryness affords crude3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid which is characterized by TLC analysis and liquid chromatography.The crude product is purified by preparative TLC using silica gel platesand a solvent system consisting of benzene 50; methanol 10; and aceticacid 6.

EXAMPLE 20 Dibenzylethylenediamine salt of3-methyl-7-methoxy-7β-(2-thienylacetamido-3-cephem-4-carboxylic Acid

Step A:

7β-(D-5-Amino-5-carboxyvaleramido)-3-methyl-7-methoxy-3-cephem-4-carboxylicAcid

A 10% palladium on charcoal catalyst is suspended in water (80 ml.) andtreated with hydrogen. The catalyst is filtered and suspended again inwater (50 ml.) and to this mixture (2.67 g.) is added the sodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (1.0 g.) in water (10 ml.). The resulting mixture is shaken fortwenty-two hours at room temperature. The catalyst is removed byfiltration and washed with water (50 ml.). The combined wash andfiltrate is concentrated to dryness to afford a 52.8% yield of7β-(D-5-amino-5-carboxyvaleramido)-3-methyl-7-methoxy-3-cephem-4-carboxylicacid (528 mg.).

Uv: λ max. 265μm; E_(lcm) ^(1%) is 100

Step B:

Dibenzylethylenediamine salt of7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicAcid

A solution of the disodium salt of7β-(D-5-amino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicacid (11.5 g.) is dissolved in water (150 ml.) and acetone (50 ml.). ThepH is adjusted to 9-9.1 with sodium hydroxide and 10 ml. of tert-butylazidoformate is added. The reaction mixture is stirred for 16 hours atroom temperature with additional sodium hydroxide being added tomaintain the pH at 9-9.1. The reaction mixture is extracted with ethylacetate (100 ml.) and the organic layer discarded. The product isprecipitated by lowering the pH to 2.5 with dilute hydrochloric acid.The precipitate is collected by centrifugation and converted to itsdibenzylethylenediamine salt which is crystallized from ethyl acetate.There is obtained 4.3 g. of the dibenzylethylenediamine salt of7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicacid, m.p. 177°-179° C. (dec.).

Uv: λmax 263μm, 238 E_(1cm) ^(1%) = 98.2, 81.1

Elemental analysis for C₃₆ H₄₉ N₅ O₉ S: Calc.: C, 59.42; H, 6.74; N,9.63; Found: C, 60.02; H, 6.80; N, 9.79.

Step C:

Dibenzylethylenediamine salt of3-methyl-7-methoxy-7β-(2-thienylacetamido-3-cephem-4-carboxylic Acid

The7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-3-methyl-7-methoxy-3-cephem-4-carboxylicacid is treated with aqueous dilute hydrochloric acid (200 ml., 0.1 N)and ethyl acetate (100 ml.) in order to extract the free acid. To asolution of 1.33 g. (2.74 mmoles) of the free acid in methylene chloride(10 ml.) is added bis-trimethylsilyl trifluoroacetamide (2.2 ml.) andmono-trimethylsilyl trifluoroacetamide (0.5 ml.). 2-Thienylacetylchloride (1.1 ml.) is then added and the reaction mixture stirred for 18hours under a nitrogen atmosphere at 43° C. The solvent is removed invacuo, and the residue partitioned between ethyl acetate and aqueousphosphate buffer (pH 7.5). The aqueous layer is acidified with dilutehydrochloric acid and the precipitated product extracted with ethylacetate. Addition of dibenzethylenediamine results in crystallization of250 mg. of the desired product as a salt in the proportion of 2equivalents of product to one mole of dibenzylethylenediamine.Recrystallization of the salt from ethanol affords substantially pureproduct, m.p. 153° -155° C. (dec.) with previous darkening.

Elemental analysis for C₄₆ H₅₂ S₄ N₆ O₁₀ : Calc.: C, 56.54; H, 5.36; N,8.60; S, 13.12; Found: C, 55.75; H, 5.16; N, 8.37; S, 12.16.

EXAMPLE 217-(Phenylacetyl-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylicAcid

A suspension of sodium cephalothin (3.36 g.) in anhydrous alcohol-freechloroform (20 ml.) is silylated by the addition oftrimethylchlorosilane (2.2 ml.). After stirring for 30 minutes,monosilyltrifluoroacetamide (5.0 ml.) and phenylacetyl chloride (4.0ml.) are added and the mixture is then heated to 45° C. for two daysunder a condenser fitted with a drying tube. The volatiles areevaporated to afford a residue which is dissolved in 100 ml. of ethylacetate and washed three times with water. The ethyl acetate layer isdried over magnesium sulfate, filtered and the solution evaporated invacuo to a residue. The residue is triturated with chloroform, anyinsolubles are removed by filtration and the product precipitated fromthe filtrate with hexane. This procedure is followed two more times. The7-(phenylacetyl-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylicacid is obtained in a solvent-free form by freeze drying from a solutionin benzene.

Ir: (chcl₃) 1780μ 1720μ

Nmr: (cdcl₃) - Consistent with structure; ##STR13## (4.03 ppm),##STR14## (4.3 ppm) TLC: 1 major spot, R_(f) = 0.69 (EtOAC:62, C₅ H₅N:21, HOAC:6, H₂ 0:11) on silica gel.

EXAMPLE 227-(Di-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic Acid

A suspension of sodium cephalothin (1.18 g.) in anhydrous alcohol-freechloroform (10 ml.) is silylated by the addition oftrimethylchlorosilane (1.1 ml.). After stirring for 30 minutes,monosilyltrifluoroacetamide (2.5 ml.) and 2-thienylacetyl chloride (2.0ml.) are added to the suspension which is then heated to 45° C. Themixture is allowed to remain at this temperature for two days and thenevaporated in vacuo to a residue which is dissolved in ethyl acetate (50ml.) and washed three times with water. The ethyl acetate layer is driedover magnesium sulfate, filtered and the filtrate evaporated in vacuo toobtain a residue. The residue is dissolved in chloroform andprecipitated with hexane three times, each time discarding thesupernatent liquid. The7-(di-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic acidis obtained in a solvent-free form by freeze-drying from a solution inbenzene.

Nmr: (solvent - CDCl₃) - Consistent with structure; β=4.25 ##STR15##TLC: 1 major spot, R_(f) =0.67 (EtOAC:62, C₅ H₅ N:21, HOAC:6, H₂ O:11)on silica gel.

EXAMPLE 237-(2-Thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic Acid

7-(Phenylacetyl-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylicacid (250 mg.) is dissolved in tetrahydrofuran (10 ml.) and water (10ml.). The pH of the solution is adjusted to 9 and the mixture is allowedto stand for one hour. After this the solution is extracted with ethylacetate and the extracts are washed with a disodium hydrogen phosphatesolution. After drying the solvent is evaporated to afford a mixture ofthe 7-(2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic acidand the starting material. The product is separated from the startingmaterial by chromatography to afford substantially pure7-(2-thienylacetyl)amino-3-acetoxymethyl-3cephem-4-carboxylic acid. Theratio of these two products is 7:3.

The cephalosporins produced by the process of this invention arevaluable antibiotics active against various gram-positive andgram-negative bacteria. Thus,3-acetoxymethyl-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid, alsoknown generically as cephalothin, is a valuable antibiotic used as ahuman therapeutic agent. The 7-methoxy cephalosporins have biologicalspectrums similar to unsubstituted cephalosporins and, in addition,possess other distinctive properties. Thus, in general, they are activeagainst many microorganisms which are resistant to the knowncephalosporins such as cephaloridine and cephalothin and are resistantto the β-lactamase produced by cephalosporin resistant clinical isolatesof pathogens such as E. coli and A. cloacae. Also, they are generallymore active against strains of Proteus such as mirabilis, and are activeagainst strains of Proteus morganii which are resistant to theunsubstituted cephalosporins. They are useful in separatingmicroorganisms in remaining susceptible microorganisms frompharmaceutical, medical and dental equipment and as bactericides inindustrial applications, for example in water-based paints and in thewhite water of paper mills to inhibit the growth of harmful bacteria.

Thus, the 7-methoxy cephalosporins produced in accordance with theprocesses of this invention are generally more active than the7-(D-5'-amino-5'-carboxyvaleramido)-7-methoxycephalosporins againstvarious gram-negative organisms and possess increased activity againstgram-positive organisms. For example, these 7-methoxy cephalosporins areactive against gram-positive pathogens such as Staphylococcus aureus atMinimum Inhibitory Concentrations (MIC) as low as about 1.5 mcg./ml.,Streptococcus pyogenes at MIC of about 0.7 mcg./ml., and Diplococcuspneumoniae at MIC of about 0.7 mcg./ml.; and against gram-negativeorganisms such as Aerobacter aerogenes at MIC of about 3 mcg./ml.Proteus vulgaris at MIC of about 1.5 mcg./ml. and Proteus morganii atabout 6 mcg./ml. Thus, activities of specific products of the foregoingexamples that might be mentioned are:3-carbamoyloxymethyl-7-methoxy-7-phenylacetamido-3-cephem-4-carboxylicacid, S. pyogenes MIC 1.56 mcg./ml. and P. vulgaris MIC 1.56 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid, S. pyogenes MIC 0.78 mcg./ml. and P. morganii MIC 12.5 mcg./ml; 3carbamoyloxymethyl-7-methoxy-7-(2-furylacetamido)-3-cephem-4-carboxylicacid, S. aureus MIC 6.25 mcg./ml. and P. vulgaris MIC 1.56 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-thiophenoxyacetamido-3-cephem-4-carboxylicacid, S. pyogenes MIC 0.78 mcg./ml. and D. pneumoniae MIC 0.78 mcg./ml.;3-acetoxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid, S. pyogenes MIC 1.56 mcg./ml. and P. vulgaris MIC 0.78 mcg./ml.;and3-pyridiniummethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid Serratia MIC 25 mcg./ml. and S. aureus MIC 156 mcg./ml.

The products of this invention may be used alone or in combination asthe active ingredient in any one of a variety of pharmaceuticalpreparations. These antibiotics and their corresponding salts may beemployed in capsule form or as tablets, powders or liquid solutions oras suspensions or elixirs. They may be administered orally,intravenously or intramuscularly. Suitable carriers which may be used inthe composition include, for example, mannitol, sucrose, glucose orsterile liquids such as water, saline, glycols and oils of a petroleum,animal, vegetable or synthetic origin as, for example, peanut oil,mineral oil, or sesame oil. Also, in addition to a carrier, the instantcompositions may include other ingredients such as stabilizers, binders,antioxidants, preservatives, lubricators, suspending agents, viscosityagents or flavoring agents and the like. In addition, there may also beincluded in the composition other active ingredients including otherantibiotics to provide a broader spectrum of antibiotic activity.

EXAMPLE 24

To a solution of 19.6 g. of ethyl urethane and 27.8 ml. of triethylaminein 300 ml. of benzene cooled to 15° C. is added dropwise 12.1 ml. ofdimethyldichlorosilane while maintaining the temperature below about 18°C. The resulting reaction mixture is allowed to stir for 15 minutes,filtered and the cake washed with a small amount of benzene. Thefiltrate and washes are concentrated to about 30 ml. in vacuo and thenfiltered and the cake washed with a small amount of benzene. Theresulting filtrate and washes are placed under reduced pressureovernight, whereupon the bis(ethoxycarbonylamino)dimethyl silane of theformula

    (CH.sub.3).sub.2 =Si=(NHCOOC.sub.2 H.sub.5).sub.2

crystallizes out and is separated and dried in vacuo.

A mixture of 100 mg. of di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-(D-5-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-cephem-4-carboxylate,135 μl. of α-pinene, 164 mg. of the dimethylsilane described above, 70μl. of thienylacetyl chloride, and 2.4 ml. of dichloroethane are heatedtogether at 65° C. for 1 hour. Chromatography of the resulting productshowed that the desired imide, di(methoxymethyl)3-carbamoyloxymethyl-7-methoxy-7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)thienylacetylamino]-3-cephem-4-carboxylate,is produced.

EXAMPLE 25

To 1 g. of di(methoxymethyl)7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyoxymetyl-7-methoxy-3-cephem-4-carboxylatedissolved in 24 ml. dichloroethane containing 1.35 ml. α-pinene is added1.7 g. of N-trimethylsilyldiphenyl phosphonamide which is convenientlyprepared from diphenyl phosphochloridate and hexamethyl disilazane.

To this mixture is added 0.7 ml. thienylacetyl chloride and thetemperature is raised to 65° C. and maintained there for 12 hours. Afterthe mixture is cooled to room temperature it is added to a solution of50 ml. of 2% NaCl-2.5% NaHCO₃ and 3 ml. of methanol is added. The twolayers are vigorously stirred for 15 minutes at room temperature.

The layers are separated and the organic layer added to ice cold hexane(250 ml.). The gum, after decantation of the hexane, is re-dissolved inmethylene chloride and the hexane precipitation repeated.Di(methoxymethyl)7β-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)thienylacetylamino-3-carbamoyloxymethyl]-7-methoxy-3-cephem-4-carboxylateso obtained is ready for further processing.

EXAMPLE 26

To 1 g. of imide methoxymethyl ester (obtained as described in Example25) dissolved in dichloroethane (10 ml.) containing 2 ml. of α-pinene isadded 0.3 ml. of trimethylsilyl chloride. The solution is heated at 65°C. for 11/2 hours under a nitrogen atmosphere. The solvent is removed invacuo and the residue is dissolved in 5 ml. of ethyl acetate. To this isadded 5 ml. of benzene and the solution is cooled to 0° C. for severalhours. The methoxymethyl ester of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid is filtered off, washed with 5 ml. cold benzene/ethyl acetate (1:1)and dried in vacuo.

EXAMPLE 27

Two grams of di(methoxymethyl)7β-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxy-methyl-7-methoxy-3-cephem-4-carboxylatedissolved in 48 ml. of dichloroethane containing 2 ml. of α-pinene istreated with 1.9 g. of N-trimethylsilyl methane sulfonamide followed by1.4 ml. of thienylacetyl chloride. The clear solution is heated at 65°C. for 12 hours under nitrogen atmosphere. After the solution is cooledto room temperature, it is added dropwise to 500 ml. ice cold hexane.The hexane is decanted from the gum and discarded. The gum isre-dissolved in 20 ml. of methylene chloride and re-precipitated using500 ml. of cold hexane. The gum, after decantation of the hexane, isdissolved in a mixture of 75 ml. ethyl acetate and 25 ml. methanol. Thesolution is cooled to 0° C., 2 ml. of concentrated HCl is added and themixture kept at 0° C. for 90 minutes. The solution is added to 52 ml.water containing 5.2 g. NaHCO₃. The bicarbonate layer is extracted with2 × 20 ml. ethyl acetate and the ethyl acetate layer is discarded. ThepH of the bicarbonate layer is adjusted to 1.8 and extracted with 3 × 25ml. ethyl acetate. On concentration of the organic layer in vacuo,3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid crystallizes out.

The cephalosporins produced by the process of this invention arevaluable antibiotics active against various gram-positive andgram-negative bacteria. Thus,3-acetoxymethyl-7β-(2-thienylacetamido)-3-cephem-4-carboxylic acid, alsoknown generically as cephalothin, is a valuable antibiotic used as ahuman therapeutic agent. The 7-methoxy cephalosporins have biologicalspectrums similar to unsubstituted cephalosporins and, in addition,possess other distinctive properties. Thus, in general, they are activeagainst many microoganisms which are resistant to the knowncephalosporins such as cephaloridine and cephalothin and are resistantto the β-lactamase produced by cephalosprin resistant clinical isolatesof pathogens such as E. coli and A. cloacae. Also, they are generallymore active against strains of Proteus such as mirabilis, and are activeagainst strains of Proteus morganii which are resistant to theunsubstituted cephalosporins. They are useful in separatingmicroorganisms in remaining susceptible microorganisms frompharmaceutical, medical and dental equipment and as bactericides inindustrial applications, for example in water-based paints and in thewhite water of paper mills to inhibit the growth of harmful bacteria.

Thus, the 7-methoxy cephalosporins produced in accordance with theprocesses of this invention are generally more active than the7-(D-5'-amino-5'-carboxyvaleramido)-7-methoxycephalosporins againstvarious gram-negative organisms and possess increased activity againstgram-positive organisms. For example, these 7-methoxy cephalosporins areactive against gram-positive pathogens such as Staphylo-coccus aureus atMinimum Inhibitory Concentrations (MIC) as low as about 1.5 mcg./ml.,Streptococcus pyogenes at MIC of about 0.7 mcg./ml., and Diplococcuspneumoniae at MIC of about 0.7 mcg./ml.; and against gram-negativeorganisms such as Aerobacter aerogenes at MIC of about 3 mcg./ml.Proteus vulgariS at MIC of about 1.5 mcg./ml. and Proteus morganii atabout 6 mcg./ml. Thus, activities of specific products of the foregoingexamples that might be mentioned are:3-carbamoyloxymethyl-7-methoxy-7-phenylacetamido-3-cephem-4-carboxylicacid, S. pyogenes MIC 1.56 mcg./ml. and P. vulgaris MIC 1.56 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid, S. pyogenes MIC 0.78 mcg./ml. and P. morganii MIC 12.5 mcg./ml;3-carbamoyloxymethyl-7-methoxy-7-(2-furylacetamido)-3-cephem-4-carboxylicacid, S. aureus MIC 6.25 mcg./ml. and P. vulgaris MIC 1.56 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-thiophenoxyacetamido-3-cephem-4-carboxylicacid, S. pyogenes MIC 0.78 mcg./ml. and D. pneumoniae MIC 0.78 mcg./ml.;3-acetoxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid, S. pyogenes MIC 1.56 mcg./ml. and P. vulgaris MIC 0.78 mcg./ml.;and3-pyridiniummethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid Serratia MIC 25 mcg./ml. and S. aureus MIC 156 mcg./ml.

The products of this invention may be used alone or in combination asthe active ingredient in any one of a variety of pharmaceuticalpreparations. These antibiotics and their corresponding salts may beemployed in capsule form or as tablets, powders or liquid solutions oras suspensions or elixirs. They may be administered orally,intravenously or intramuscularly. Suitable carriers which may be used inthe composition include, for example, mannitol, sucrose, glucose orsterile liquids such as water, saline, glycols and oils of a petroleum,animal, vegetable or synthetic origin as, for example, peanut oil,mineral oil, or sesame oil. Also, in addition to a carrier, the instantcompositions may include other ingredients such as stabilizers, binders,antioxidants, preservatives, lubricators, suspending agents, viscosityagents or flavoring agents and the like. In addition, there may also beincluded in the composition other active ingredients including otherantibiotics to provide a broader spectrum of antibiotic activity.

Although the salts of cephalosporins are generally used as therapeuticagents, certain esters of these cephalosporins are also especiallyuseful for human and animal therapy. Especially preferred are thoseesters which are converted biologically to the free acid or a saltthereof. Examples of such esters that might be mentioned areloweralkyloxy loweralkyl esters such as methoxymethyl, methoxyethyl, andthe like; aralkyloxy loweralkyl esters such as benzyloxymethyl,benzyloxyethyl, phenoxyethyl, and the like; and acyloxy loweralkylesters such as the acetoxymethyl, pivaloyloxymethyl, pivaloyloxyethyl,propionyloxymethyl, butyryloxymethyl, isobutyryloxymethyl,benzoyloxymethyl ester and the like. The term "loweralkyl" refers tostraight or branched chain alkyl groups having 1 to 5 carbon atoms.These esters can be prepared directly by using the desired ester in theabove-described processes and recovering the ester of the desiredcephalosporin compound. For example, when the methoxymethyl ester of the7β-adipoyl cephalosporin compound is used in the above-describedtransacylation process, the methoxymethyl ester of the new7β-acylamidocephalosporin compound is produced directly as is shown inthe foregoing examples. Alternatively, the cephalosporin free acidsproduced by the processes of this invention are readily esterified byreaction with the appropriate haloloweralkyl moiety in accordance withmethods well known in the art. Thus, for example, the methoxymethylester and the phenoxymethyl ester are readily prepared by reactingchloromethyl methyl ether or α-chloroanisole with the cephalosporin acidin the presence of an acid scavenger, or by reacting a salt of thecephalosporin acid with the halo compound. Similarly, theacyloxyloweralkyl esters are readily prepared by reacting thecephalosporin acid or a salt thereof with the chloro or bromomethylester of the appropriate acid pursuant to methods known in the art.

We claim:
 1. The process for preparing the compound of the formula:##STR16## wherein R₁ is hydrogen or methoxy; A is loweralkanoyloxy of2-6 carbon atoms or carbamoyloxy;R₁₀ is hydrogen, azido, carboxy, orhydroxy; R₁₁ is phenyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,3-isothiazoyl, 4-isothiazoyl, 5-isothiazoyl, 1,2,5-thiadiazoyl,4-pyridyl, tetrazoyl, or chlorophenyl; X is oxygen or sulfur; and R₄ ishydrogen, or methyl, ethyl, tertiary butyl, phthalimidomethyl,succinimidomethyl, phenacyl, p-bromophenacyl, 2,2,2-trichloroethyl,2-methylthioethyl, 2-(p-methylphenyl)ethyl,2-(p-methylphenyl)-sulfonylethyl, 2-methylaminoethyl, 2-chloro(orbromo)ethyl, benzyl, p-nitrobenzyl, p-methoxybenzyl, 3,5-dinitrobenzyl,2,4,6-trimethylbenzyl, 3,5-dichloro-4-hydroxybenzyl, benzhydryl,p-methoxybenzhydryl, phenyl, acetoxymethyl, pivaloyloxymethyl,methoxymethyl, p-nitrophenyl or 3,5-dinitrophenyl; which comprisesmixing the compound of the formula: ##STR17## in which R₂ istrichloroethoxycarbonyl, tertiary butoxy carbonyl,benzoylmethoxycarbonyl, trimethylsilyl, p-methoxybenzyloxy,2-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl, chloroacetyl, oro-nitrophenylthio; and R₁, A, X and R₄ are as above defined, with anapproximately equimolar amount of the acylating agent having the formula##STR18## where R₁₁ and R₁₀ are as defined above and with a molecularexcess of the silylating agent which is N-(trimethylsilyl)acetamide,N-(tripropylsilyl)acetamide, N-(tributylsilyl)acetamide,N-(triphenylsilyl)acetamide or N-(tribenzylsilyl)acetamide,N,O-bis(trimethylsilyl)acetamide, N,O-bis(triphenylsilyl)acetamide,N,O-bis(tribenzylsilyl)acetamide, N-(trimethylsilyl)trifluoroacetamide,N-(tributylsilyl)trifluoroacetamide, N-(trimethylsilyl)benzoamide,N-(trimethylsilyl)diphenylurea, N-(trimethylsilyl)ethylcarbamate,N-triphenylsuccinimide or N-(trimethylsilyl)phthalimide,N-(trimethylsilyl)benzene sulfonamide, N-trimethylsilyl urethane,N-trimethylsilyl phthalimide, monosilyltrifluoroacetamide orbis(ethoxycarbonylamino)dimethyl silane in an inert solvent at atemperature of between about 25° C and about 70° C. for a duration of3-65 hours, and then optionally adding loweralkanol or loweralkylthiol,alkyl having 1-6 carbon atoms benzylalcohol, 2.5N HCl or aqueous sodiumbicarbonate; and recovering the product.
 2. The process of claim 1 inwhich the inert solvent is acetonitrile, chloroform, dichloromethane, ormethylene chloride.
 3. The process of claim 1 in which R₁ is methoxy. 4.The process of claim 3 in which A is carbamoyloxy.
 5. The process ofclaim 3 in which A is loweralkanoyloxy of 2-6 carbon atoms.
 6. Theprocess of claim 5 in which A is acetoxy.
 7. The process for preparingthe compound ##STR19## in which A is acetoxy or carbamoyloxy, and R₁₁ isphenyl, 2-thienyl, 3-thienyl, 2-furyl, or 3furyl, and R₅ is H,methoxymethyl, benzyl, or benzhydryl, which comprises mixingapproximately equimolar amounts of ##STR20## in which R₂ istrichloroethoxycarbonyl, t-butoxycarbonyl, chloroacetyl, or2-nitrophenylsulfenyl, and A and R₅ are defined above; and ##STR21##together with a molecular excess of N-trimethylsilyl urethane,bis-trimethylsilyltrifluoroacetamide, N-trimethylsilyl phthalimide,N-trimethylsilylethyl carbamate, N-trimethyllsilylbenzene sulfonamide,or monosilyltrifluoroacetamide, in an inert solvent which isacetonitrile, chloroform, dichloromethane, or methylene chloride, at atemperature between 25° C. and about 70° C., for about 3 - 65 hours,then optionally adding loweralkanol, benzyl alcohol, 2.5N HCl or aqueoussodium bicarbonate, and recovering the product.